Question

The lab in which you work wants to use a yeast two-hybrid (Y2H) assay to identify binding partners (prey) for a newly discovered yeast protein (bait). Your lab's Y2H system uses the Gal4 transcription factor. You begin by testing the Y2H system with a pair of proteins that exhibit a strong, direct binding interaction in several in vitro binding assays. Unfortunately, you do not see any surviving colonies. You know that the commercial Y2H system you used in your experiment is reliable and has been shown to work with bait and prey‑positive controls.

Choose the statements that are reasonable explanations for the failure of your Y2H experiment.

The expression levels of the fused bait or prey proteins were too low.

Fusing the bait or prey to portions of Gal4 resulted in loss of bait and prey binding due to steric hindrance.

The Y2H system lacked necessary cofactors or allosteric effectors required for bait and prey binding.

One or both of the fusion proteins did not localize to the nucleus.

The Gal4 DNA‑binding domain did not bind to its target DNA sequence.

Answer 1

The expression levels of the fused bait or prey proteins were too low. Even at low expression levels, Y2H is known to work, so this reason should not be at the top of the list of possibilities.

Fusing the bait or prey to portions of Gal4 resulted in loss of bait and prey binding due to steric hindrance. If the bait and/or prey do not fuse in the correct orientation, the DNA-binding region and the activating domain will not be able to bind and subsequently activate the transcription factor.

The Y2H system lacked necessary cofactors or allosteric effectors required for bait and prey binding. This is plausible. The expression of the interacting proteins will not be possible and hence no colonies will be seen.

One or both of the fusion proteins did not localize to the nucleus. This is not a plausible cause if the commercial system used is reliable since the vector used for expression as the proteins itself contains a nuclear localization signal. However, if for any reason, nuclear localization does not take place, no colonies will be detected.

The Gal4 DNA-binding domain did not bind to its target DNA sequence. This is also not possible if the commercial system is reliable. If the proteins interact, then the target DNA binding depends on the efficiency of the system, not the interacting proteins.